Modification Of Foot And Mouth Disease Virus Leader Protein Deleted Infectious Clone And Virus Rescue

Foot and mouth disease(FMD) is one of the most important diseases of domestic cloven-hoofedanimals such as swine, bovine and sheep. The different measures have been taken to control anderadicate FMD in the world. The vaccination is considered to be a practicable strategy in FMD epidemicarea, however, the safe and efficient vaccine is the key of this strategy and it also is an aim forresearchers.In our laboratory, an infectious cDNA clone from a type O FMDV vaccine strain (OZK/93) wassuccessfully constructed and then the gene coding leader-proteinase was deleted, from which anengineering attenuated FMDVstrain was obtained. In order to use the attenuated strain infectious cDNAto develop new vaccine, two different strategies have been applied as following:Firstly, the extrinsic gene, coding enhance fluorescence protein (eGFP) linking with 2A gene wasinserted between non-structure protein 2Aand 2B in the infectious cDNAclone p43-CHA90-LL, namedas p43-CHA90-LL-eGFP2A. The plasmid DNA or in vitro transcripted RNA was transfected intoBHK21 cell using lipofectamine reagent. The green fluorescence was observed in transfected cellsunder fluorescence microscope. The result indicated that eGFP was successfully expressed. After 48hours the transfected cells were harvested and passaged in new monolayer BHK21 cell. Thecytopathogenic effect (CPE) was observed, but the green fluorescence was disappearing. The resultshowed that the live virus was rescued from p43-CHA90-LL-eGFP2A, but the extrinsic gene did notexpress eGFP in progeny virus. PCR and sequencing results showed that the eGFP gene was completelydeleted from rescued virus. Although eGFP can translate in infectious cDNA clone, its gene couldunstably pass to offspring virus in which the extrinsic gene was deleted by an unknown mechanismduring viral RNAreplication or virus assembly. All those results prove that the gate is closed to developbivalent vaccine or combined vaccine by inserting extrinsic gene between 2A and 2B in FMDVinfectious cDNAuntil we know the mechanismthat how the extrinsic gene was deleted.Secondly, based on the FMDV capsid protein composing and three-dimensional structurecharacteristic, a cloning vector was constructed by silent mutation replacing structural protein VP1, VP2and VP3 with multiple cloning site SspI and SgrAI from Leader protein deleted attenuated straininfection cDNA. In this study, VP1, VP2 and VP3 coding sequence of O type FMDV prevalent strainfrom swine was successfully inserted into vector. The chimeric virus was rescued in BHK21 cell. Thisgenetically engineering attenuated strain can be used as a candidate of safe vaccine strain, due toincluding part of prevalent strain genome and express same immunogenicity of prevalent strain.